ORIGINAL RESEARCH published: 05 January 2016 doi: 10.3389/fmicb.2015.01486

Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem

Jorge A. Valdivia-Anistro1, Luis E. Eguiarte-Fruns1, Gabriela Delgado-Sapién2, Pedro Márquez-Zacarías3, Jaime Gasca-Pineda1, Jennifer Learned4, James J. Elser4, Gabriela Olmedo-Alvarez5 and Valeria Souza1*

1 Laboratorio de Evolución Molecular y Experimental, Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Coyoacán, Mexico, 2 Laboratorio de Genómica Bacteriana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Coyoacán, Mexico, 3 School of Biology, Georgia Institute of Technology, Atlanta, GA, USA, 4 School of Life Sciences, Arizona State University, Tempe, AZ, USA, 5 Laboratorio de Bacteriología Molecular, Departamento de Ingeniería Genética, CINVESTAV – Unidad Irapuato, Irapuato, Mexico

The ribosomal RNA (rrn) operon is a key suite of genes related to the production

Edited by: of protein synthesis machinery and thus to bacterial growth physiology. Experimental Roland Hatzenpichler, evidence has suggested an intrinsic relationship between the number of copies of this California Institute of Technology, USA operon and environmental resource availability, especially the availability of phosphorus Reviewed by: (P), because bacteria that live in oligotrophic ecosystems usually have few rrn operons Sebastian Kopf, Princeton University, USA and a slow growth rate. The Cuatro Ciénegas Basin (CCB) is a complex aquatic Albert Leopold Mueller, ecosystem that contains an unusually high microbial diversity that is able to persist Stanford University, USA under highly oligotrophic conditions. These environmental conditions impose a variety *Correspondence: Valeria Souza of strong selective pressures that shape the genome dynamics of their inhabitants. The [email protected] genus Bacillus is one of the most abundant cultivable bacterial groups in the CCB and usually possesses a relatively large number of rrn operon copies (6–15 copies). The Specialty section: This article was submitted to main goal of this study was to analyze the variation in the number of rrn operon copies Microbial Physiology and Metabolism, of Bacillus in the CCB and to assess their growth-related properties as well as their a section of the journal Frontiers in Microbiology stoichiometric balance (N and P content). We defined 18 phylogenetic groups within the Received: 16 September 2015 Bacilli clade and documented a range of from six to 14 copies of the rrn operon. The Accepted: 09 December 2015 growth dynamic of these Bacilli was heterogeneous and did not show a direct relation Published: 05 January 2016 to the number of operon copies. Physiologically, our results were not consistent with Citation: the Growth Rate Hypothesis, since the copies of the rrn operon were decoupled from Valdivia-Anistro JA, Eguiarte-Fruns LE, growth rate. However, we speculate that the diversity of the growth properties of these Delgado-Sapién G, Bacilli as well as the low P content of their cells in an ample range of rrn copy number Márquez-Zacarías P, Gasca-Pineda J, Learned J, Elser JJ, is an adaptive response to oligotrophy of the CCB and could represent an ecological Olmedo-Alvarez G and Souza V mechanism that allows these taxa to coexist. These findings increase the knowledge (2016) Variability of rRNA Operon of the variability in the number of copies of the rrn operon in the genus Bacillus and Copy Number and Growth Rate Dynamics of Bacillus Isolated from an give insights about the physiology of this bacterial group under extreme oligotrophic Extremely Oligotrophic Aquatic conditions. Ecosystem. Front. Microbiol. 6:1486. doi: 10.3389/fmicb.2015.01486 Keywords: rRNA operon copies, oligotrophy, bacterial growth, Bacillus, Cuatro Ciénegas

Frontiers in Microbiology | www.frontiersin.org 1 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

INTRODUCTION copy number may be favored because the rich P supply could then support the rapid production of P-rich rrn to meet the Population genetics is the most direct tool for use in protein demands of rapid growth, as stated by the “Growth understanding adaptation to the ecological challenges imposed Rate Hypothesis” (GRH), a core idea within the theory of upon microbial communities by the environment (Whitaker biological stoichiometry (Elser et al., 2000; Elser, 2006). However, et al., 2003; Xu, 2006; Spencer et al., 2008). Functional traits can in environments with low phosphorus availability, multiple aid in the study of population genetics, because they help to define rrn operon copies could represent a competitive cost if a species in terms of their ecological roles, such as how they use high rrn operon copy number leads to the over-production environmental resources or how they interact with other species of P-rich rrn (Sterner and Elser, 2002; Maciá, 2005; Jeyasingh (McGill et al., 2006; Hughes et al., 2008). These functional traits and Weider, 2007). Indeed, aquatic bacteria isolated from are often considered to be ecological strategies because they are oligotrophic environments usually have low rrn operon copy useful in understanding why certain bacteria live in a particular numbers (Fegatella et al., 1998; Strehl et al., 1999; Lauro et al., environment and how they respond to environmental challenges 2009), as well as various other adaptations to decrease cellular (Green et al., 2008). phosphorus demand (Cavicchioli et al., 2003; Alcaraz et al., The ribosomal RNA operon (rrn hereafter) is the key genetic 2008; Martiny et al., 2009; Van Mooy et al., 2009). Thus, it has structure for protein synthesis and thus a functional trait been proposed that there is a connection between the number related to bacterial life history (Stevenson and Schmidt, 2004). of rrn operon copies and environmental P availability (Elser Ecologically, the rrn operon has been related with the bacterial et al., 2000; Weider et al., 2005; Jeyasingh and Weider, 2007). capacity to respond to changes in environmental conditions However, to our knowledge, we lack extensive studies that (Codon et al., 1995; Prüβ et al., 1999; Green et al., 2008). In document this variation in rrn operon copy number and other particular, the variation in the number of copies of the rrn associated ecological strategies employed by bacteria coexisting operon has been considered an ecological strategy related to in oligotrophic environments, especially those characterized by resource availability, with physiological implications associated severe P limitation. with bacterial growth rate and fitness (Klappenbach et al., 2000; The aims of this study were to describe rrn operon Shrestha et al., 2007). The rrn operon is comprised of three genes copy number variation in different lineages of Bacillus strains (5S, 16S, and 23S rDNA) and its copy number varies from 1 to isolated from an extremely oligotrophic ecosystem and to 15 among bacterial genomes (Klappenbach et al., 2001; Acinas analyze the possible association between the copy number and et al., 2004; Stoddard et al., 2015) and even more dramatically its physiological implications for growth rate and chemical among eukaryotes (Elser et al., 2000). Experimentally, it has been composition (P content and N:P stoichiometry). Severe P shown that deletions of one or more copies of the rrn operon limitation is considered a primary selective pressure that drives have a considerable impact on growth rate, affecting various bacterial evolution in this environment (Souza et al., 2008, 2012). stress-response mechanisms (Nanamiya et al., 2010; Yano et al., For example, we have previously reported on an endemic and 2013). Hence, it has been suggested that the multiplicity of the moderately halophilic Bacillus (type strain of B. coahuilensis: rrn operon is a potential mechanism for adaptation to different m4-4 = NRRL B-41737T), (Cerritos et al., 2008)thathasa environmental conditions (Elser et al., 2000; Green et al., 2008). typical number of rrn copies (nine) but also clear adaptations to In general terms, bacteria that possess more rrn operon copies the extreme oligotrophic conditions, including a small genome may cope better with fluctuating nutrient inputs than bacteria (3.5 Mb), a diversity of phosphate acquisition genes (Moreno- with fewer rrn operon copies, which tend to live in environments Letelier et al., 2011), and a cellular membrane composed of where nutrients are scarce (Klappenbach et al., 2001; Elser, 2003; sulfolipids (Alcaraz et al., 2008). Similar adaptations to low Jeyasingh and Weider, 2007). Moreover, the relationship between P levels have been reported only from oligotrophic marine rrn operon copy number and the bacterial biotic potential for cyanobacteria with low rrn copy numbers (Cavicchioli et al., the cellular allocation of key resources could be analogous to 2003; Lauro et al., 2009; Martiny et al., 2009; Van Mooy et al., the ecological strategies described in other macro-biota (r- and 2009). Hence, the present study represents the first attempt to link K-strategies), (Pianka, 1970; Elser et al., 2000; Dethlefsen and biological stoichiometry to Bacillus diversity and rrn operon copy Schmidt, 2007; Shrestha et al., 2007; Lipowsky et al., 2012). number, as well as the first report in which the numbers of rrn Bacillus is a genus that is well-known because of its copies are analyzed in several members of this genus that coexist ecological versatility (Feldgarden et al., 2003). The fact that in the same habitats. it can sporulate increases its long-range dispersal and allows it to explore diverse environments (Nanamiya et al., 2010; Yano et al., 2013). Coincidentally, Bacilli have a relatively MATERIALS AND METHODS high number of rrn operon copies per genome, ranging from six to 15 (rrnDB, Stoddard et al., 2015), a degree of Site Description and Selection of variation that may aid in this lifestyle strategy of colonizing Bacillus Strains new environments and provide great adaptability in response The Cuatro Ciénegas Basin (CCB hereafter) is a hydrologic to stress, as well as being able to uptake a wide variety of system in the Chihuahuan Desert in northeastern México (Souza resources (Feldgarden et al., 2003; Connor et al., 2010). If the et al., 2006), (Figure 1A, yellow triangle). This basin represents new environment is rich in phosphorus (P), high rrn operon an oasis with a high microbial diversity in extremely oligotrophic

Frontiers in Microbiology | www.frontiersin.org 2 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

FIGURE 1 | The Cuatro Ciénegas Basin (CCB) in the Chihuahuan Desert, in northeastern México (A,B) and the sites where Bacillus strains were previously isolated. (C) The Churince system, (D) Pozas Rojas (Los Hundidos), and (E) Río Mesquites. (F) Maximum-Likelihood (ML) tree of the 19 phylogenetic groups identified using the 5 HV region of the 16S rDNA. Bootstrap values higher than 70% are shown. Symbols represent the sample type of isolation:  = Top section of sediment,  = Bottom section of sediment,  = Water sediment adjacent to a plant, and  = Water.

Frontiers in Microbiology | www.frontiersin.org 3 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

3− conditions (<1 μmol PO4 ; Peimbert et al., 2012; Souza et al., fragments usually representing the number of rrn operons. 2012). Interestingly, ca. 50% of the bacterial communities in the Pulsed-field gel electrophoresis (PFGE) was used to construct the CCB are most closely related to marine relatives (Souza et al., rrn profile of the chromosome from the Bacillus isolates. Bacterial 2006). Isolates related to the genus Bacillus were identified from genomic DNA from Salmonella enterica serovar Typhimurium samples collected at various sites in the CCB during 15 years of LT2 cleaved with CeuI and the 0.1–200 kb Sigma Plus Marker field work (Souza et al., 2006; Alcaraz et al., 2008, 2010; Cerritos were used as molecular weight markers. Because the size of et al., 2010; Pérez-Gutiérrez et al., 2013), (Figure 1B). S. enterica Typhimurium LT2 identifying fragments had been Our isolates are from three primary sampling sites within determined previously (Liu et al., 1993), their inclusion improved thebasin:(i)theChurincesiteconsistsofafreshwaterspring the precision of the band-size estimation. that connects to an intermediate shallow pond via a small stream and eventually terminates in a shallow desiccated lagoon Preparation and Digestion of Genomic (Figure 1C); (ii) the Río Mesquites is a stable system composed DNA in Agarose Blocks of a river and some lateral desiccated ponds (Figure 1D)with Bacillus isolates were cultured aerobically in DifcoTM Marine low nutrient concentrations and highly imbalanced C:N:P ratios Broth 2216 (BD & Co.) and incubated overnight at 35◦C. The [C:N:P, 900:150:1 (molar); Souza et al., 2012]; and (iii) the Pozas genomic DNA of each strain was prepared in agarose blocks Rojas site (Figure 1E) is located in a system called Los Hundidos using a previously described method, with some modifications and consists of a shallow lake and nine to 12 small semi- (Nakasone et al., 2000; Delgado et al., 2013). Two processes of permanent ponds with strongly fluctuating conditions (high incubation in proteinase K solution (12 h at 37◦C) were carried salinity and temperature in summer, both decrease in winter). out to increase the purity of the DNA. Agarose blocks were pre- The Bacillus strains isolated from the CCB are part of a incubated in 1X NEBuffer 4 (New England Biolabs) for 30 min at ◦ larger collection (several thousands of isolates) of microbes that 4 C. Finally, the digestion of the genomic DNA was achieved with is maintained at the Molecular Evolution and Experimental 100 μl fresh 1X NEBuffer 4 containing 15 U of I-CeuI restriction ◦ Laboratory at the Instituto de Ecología, UNAM (Valeria enzyme, and it was incubated overnight at 37 C. Souza) and at the Molecular Bacteriology Laboratory in the CINVESTAV, Irapuato (Gabriela Olmedo); cultures are available Pulsed-Field Gel Electrophoresis (PFGE) upon request. We selected 71 of these isolates and classified them and DNA Fragments Transfer according to the site of isolation and sample type (plant root, The CeuI fragments were separated by a CHEF-DR II sediment, or water). Sixty-seven Bacillus isolates were sampled electrophoresis system (Bio-Rad). Electrophoresis was performed from Churince, one was from Río Mesquites and three were from on a 1% agarose (Seakem Gold agarose, BioWhittaker Molecular Pozas Rojas. ◦ Applications) gel and 0.5X TBE buffer (Bio-Rad) at 11 C. The electrophoresis conditions were divided into two stages to Phylogenetic Analysis separate the DNA fragments of various sizes: First stage, pulse To obtain biomass for DNA extraction, Bacillus isolates were time ramped from 6.75 s to 2 min for 20 h at 4 V cm−1 and in grown in the standard medium used for their isolation in the field a second stage, pulse time ramped from 0.22 to 5.10 s for 15 h at (Marine agar, DifcoTM 2216). Genomic DNA extractions were −1 R 6Vcm . performed using the QIAmp DNA Mini Kit (USA), according TheagarosegelswereradiatedwithUVlightfor1minina to the manufacturer’s instructions. The 5 hypervariant (HV) UV Crosslinker (UVP) to fix the DNA. The gels were washed region of the 16S rDNA was amplified (275 bp; 70–344 position), in 250 mM HCl solution for 15 min with moderate shaking. following Goto et al., (2000). This region has a high level of Next, the gels were washed in denaturing buffer (1.5 M NaCl, conservation and is useful for the clustering of Bacillus species. 0.5 M NaOH) for 2 h and later washed in a neutralization The polymerase chain reaction (PCR) products were confirmed buffer (0.5 M Tris/HCl, 1.5 M NaCl; pH 8.0) for 2 h. The via 1.5% agarose gel electrophoresis. The sequencing of the HV DNA fragments were then transferred onto N+nylon membrane region was performed by the High Throughput Genomics Center (Amersham Biosciences) via Southern blotting as described (htSEQ), University of Washington (USA), and compared with previously (Sambrook et al., 1989). Finally, the membrane was the GenBank database using BLAST (NCBI). Sequences were radiated with UV light to fix the DNA (1 min; UV Crosslinker, aligned using CLUSTAL W (Thompson et al., 2002), and a UVP). maximum-likelihood tree was constructed using MEGA5, with a bootstrap of 1000 replicates (Tamura et al., 2011). The sequences Preparation of DNA Probes and of the 5 HV region of the 16S rDNA were submitted to GenBank Hybridization with the following accession numbers: KT781592–KT781661. The rrn profiles of Bacillus isolates were validated by probing the CeuI Bacillus Southern blotting membranes with PCR products of the 16S and I- Cleavage Map of the 23S rrn operon from the Bacillus horikoshii ATCC 700161 strain. Strains Theprimersetsusedtoamplifytherrs gene were designed using The I-CeuI(CeuI hereafter) restriction endonuclease recognizes the 5 HV region (described above), and an internal region of the a 26-bp sequence from position 1911–1936 of the 23S rRNA gene rrl gene was designed from the 2283 to 2696 position (23S3)of in rrn operons, with the number of CeuI (New England Biolabs) the rrn. Then, the 23S3 region (413 bp) was amplified using the

Frontiers in Microbiology | www.frontiersin.org 4 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

μ forward primer F23S3 5 -ACG GAG GCG CCC AAA GGT T- we estimated the maximum specific growth rate ( max;units: −1 3 and the reverse primer R23S3 5 -CCA GCG GTG CGT CCA hours ) as follows: TCC-3 . The primer set used to amplify the 23S ,wasdesigned 3 μ = ( − )/( − ) based on previously sequenced genomes using the Primer Select max ln Ne N0 te t0 program of the DNASTAR Lasergene 7 package (DNASTAR, Inc., and the bacterial doubling (generation) time as follows: Madison, WI, USA). ◦ The PCR amplification conditions were as follows: 95 Cfor = /μ td ln 2 max 5 min for the initial denaturation, 30 cycles of denaturation at ◦ ◦ 95 C for 40 s, annealing at 60 C for 40 s, an extension of 1 min N0 and Ne are the cell densities reached at the beginning and at ◦ ◦ at 72 C and a final extension of 5 min at 72 C(GeneAmp, the end of the exponential phase, respectively, while t0 and te are PCR System 9700). The presence and size of PCR products were the times (h) at which the exponential phase started and ended, subsequently confirmed via 1.5% agarose gel electrophoresis. respectively. The PCR products were purified with the PCR Clean-up Gel Extraction Kit (Macherey–Nagel products) and then DIG-labeled Cell Contents of Carbon (C), Nitrogen using the random primer method of the DIG High Prime DNA (N), and Phosphorus (P) During the Labeling system (Roche). The membrane was incubated in 10 ml Exponential Phase hybridization solution (DIG Easy Hyb buffer). Incubation was ◦ The samples of bacterial biomass were harvested in the carried out at 58 C with constant, gentle shaking for 1 h. The exponential phase during the determination of growth dynamics. labeled probe was then added to fresh hybridization solution and ◦ Biomass samples were spin in a centrifuge for the removal hybridization was carried out overnight at 58 Cwithconstant of the growth medium. To avoid the influence of remains of and gentle shaking. The membrane was exposed to X-ray film ◦ the growth medium in the elemental composition analysis, the after being washed at high astringency (64 C). biomass samples were washed three times with 250 μlwater R Growth Parameter Estimations (Mili-Q) and after of each washing, supernatant was removed by centrifuge. Finally, the biomass samples were vacuum dried to Genotypes of Bacillus with different numbers of copies of the be shipped frozen to Arizona State University for analysis. rrn operon were chosen from among the groups described in At ASU total phosphorus content was measured using a the phylogenetic analysis. Prior to growth parameter estimation, modified ascorbic acid method with persulfate digestion (APHA, all the strains were pre-cultured in fresh marine broth for 24 h 2005). The dried biomass samples were weighed and treated to homogenize their metabolic condition. All cultures were ◦ with a potassium persulfate and sulfuric acid solution and then incubated at 35 C, the maximum water temperature during ◦ autoclaved for 30 min at 121 C and 15–20 psi. The samples summer at the CCB (Pérez-Gutiérrez et al., 2013), with shaking were allowed to cool and then neutralized before the addition of at 150 rpm. Additionally, experiments were carried out for the color reagent. After 30 min, the absorbance was read on a nutritional conditions similar to CCB; for this, we inoculated the spectrophotometer at 880 nm. The samples were analyzed with a strains into sterile water collected from the Churince field site but triplicate standard curve and triplicate NIST reference material. supplemented with tryptone (5 g per liter; BactoTM Tryptone, The total carbon and nitrogen content was measured via BD and Company; hereafter, CCBwt) and incubated for 12 h combustion in a Perkin Elmer model 2400 elemental analyzer. (overnight), (Supplementary Figure S1A). The samples were combusted at 1760◦C. Elemental detection was The growth parameters were then determined using the conducted via a thermal conductivity detector. previously described overnight culture. Three new 50 ml flask of The C, N, and P data were expressed as percentages of dry fresh CCBwt medium were inoculated to reach an optical density mass and referred to as “C content,” “N content,” and “P content,” of 0.05 (600 nm wavelength; BioPhotometer Plus, Eppendorf), − respectively (Supplementary Table S2). which corresponded to ∼107 colony-forming units (CFU) ml 1. CFU counts were made taking at least seven samples distributed through a period of 12 h to cover all the phases of the growth Statistical Analysis curve, the samples were diluted appropriately in 0.85% NaCl to All statistical analyses, including the estimation of Pearson perform a plate count analysis. correlation coefficients (r) and principal component analysis We estimated the lag phase period (λ; units: hours), the (PCA) were performed with the R Statistic program Version 3.3.1 (24-07-10). tangential growth rate (Gtan; units: cells/h), and maximal biomass reached [A; units: Ln(CFU/CFU(t = 0))] from our data using a non-linear regression (CurveExpert Professional 2.0.3 software) to fit a Gompertz equation according to Zwietering et al. (1990), RESULTS (Supplementary Figure S1B). We obtained the final parameters Identification and Clustering of Bacillus from the predicted curve, defining Gtan as the tangent of the inflection point of the curve, λ as the X intercept of the Isolates from the CCB tangential line through the inflection point (where the X-axis The 71 isolates used for this study were clustered into 19 is time), and A as the Y-value of the asymptote [where the phylogenetic groups, forming a large “marine” cluster (30 isolates; Y-axis is Ln(CFU/CFU(t = 0))] for plate-count assays. In addition, i.e., formed by strains with marine affinities). Most of the lineages

Frontiers in Microbiology | www.frontiersin.org 5 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics include representatives from a variety of habitats (e.g., soil, water, samples; B. idriensis isolates (group XVI), were related to strains sediment); however, some “marine” groups included strains only from soil samples with halotolerance. Finally, the B. horikoshii sampled from water, while the B. atrophaeus lineage was only cluster (group XVII) has as a representative a strain isolated found in the top layers of sediment. Strains related to B. pumilus from a fish pond as well as a lineage related to a halotolerant were also only found in sediment (Figure 1F). B. nanhaiensis (group XVIII), isolated from a non-saline soil The “marine” cluster is composed of nine phylogenetic groups sample. Unexpectedly, the 169A 5R isolate was closely related (arbitrarily numbered I to IX) composed of Bacillus strains to the strain Staphylococcus sp. Mg48 (JQ399818), isolated from isolated mostly from CCB aquatic samples, such as B. sp. m2- a saline lake (group XIX), but it is not unusual to isolate a 34 (group II) and B. coahuilensis (group V). In addition, three Staphylococcus strain when aiming to select Bacillus. CCB phylogenetic groups of Bacillus were related to type strains isolated from water: B. endophyticus (group III), related to a Phylogenetic Variability of the rrn Operon pollutant-degrading strain isolated from industrial effluent, as Copy Number well as B. sp. NRRLB-14911 (group IV) and B. marisflavi (group The rrn operon copy number was determined for every isolate IX), both isolated from seawater. The B. aquamaris (group I), B. described above in the maximum-likelihood tree via PFGE and sp.SP61(groupVI),andB. vietnamensis (group VIII) groups hybridization analyses (Figure 2 and Supplementary Figure are related to strains isolated from hypersaline environments S2). To obtain a benchmark for the rrn copy number in the (salterns and a microbial mat). Finally, group VII is related Bacillus diversity from the CCB, we analyzed the type strain to a strain isolated from a biofilm of a lake (B. sp. BA-117). of B. coahuilensis (m4-4 = NRRL B-41737T) that was isolated The small B. cereus cluster is related to type strains isolated from the Churince site (Cerritos et al., 2008)andthathas from marine sediments (group X). The B. subtilis cluster (group already been sequenced (Alcaraz et al., 2008). After genomic XIII), was similar to soil type Bacillus. Group XI is most closely digestion and hybridization analysis, eight rrn operons were related to B. sonorensis isolated from the soil of the Sonoran quantified (Figure 2 and Supplementary Figure S2; group desert. Organisms in group XII are related to a B. atrophaeus V). The 70 strains of Bacillus from the CCB showed a range strain isolated from soil and water samples, and group XIII is of between six and 14 rrn operon copies (Figure 2 and related to a B. subtilis strain from marine samples, although Supplementary Figure S2). Some groups showed intraspecific this is a well-known cosmopolitan species. Strains related to a variation from one to four copies. Interestingly, we quantified B. altitudinis strain (group XV), were present in soil and water only six copies of this operon in some phylogenetic groups, such

FIGURE 2 | Variability of the rRNA operon copy number in the Bacillus diversity from the CCB. ML tree of the 5 HV region of the 16S rDNA with a 50% of cut-off value. The black circles represent the type strain of Bacillus for every phylogenetic group. The squares, triangles and rhomboids symbols correspond to the environment as described in Figure 1. The dotted red line represents the low number of the rrn operon in Bacillus according to the rrnDB. The black bars represent the number of operon copies in the isolates from the CCB, and the green bars represent the number of copies in Bacillus species reported in the rrnDB. Gray bar represents the number of copies in the isolate related to the genus Staphylococcus. The light purple bars represent the number of operon copies in different Staphylococcus species analyzed by the rrnDB. The orange bar represents the number of copies quantified in the type strain of B. coahuilensis (m4-4 = NRRL B-41737T), group V. The yellow bars represent the number of copies in the type strains of B. marisflavi (JCM 11544 = KCCM 41588) and B. horikoshii (ATTCC 700161), groups IX and XVII, respectively.

Frontiers in Microbiology | www.frontiersin.org 6 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics as B. sonorensis (XI), B. atrophaeus (XII), and B. nanhaiensis TABLE 1 | Growth parameters estimated in the Bacillus isolates in the (XVIII); the lowest number of copies quantified in other strains CCB. of the genus (rrnDB, Stoddard et al., 2015). The highest Growth parameters number of copies was observed in the B. cereus group (X) (14 rrn λ μ −1 t copies). Isolate Phylogenetic group A (h) max(h ) d(h) To further increase knowledge about the number of copies copies of the rrn operon in the genus Bacillus,weanalyzedtwotype A 14 155B_5T X. B. cereus 9.98 0.92 0.26 2.66 strains similar to those observed in the CCB, B. marisflavi (JCM B 6 118_4C XI. B. sonorensis 9.89 1.13 0.24 2.88 11544 = KCCM 41588), and B. horikoshii (ATTCC 700161). C 8 m3-18 II. B. sp. m2-34 9.78 2.89 0.24 2.88 The genomes of these species have not yet been sequenced and D9m2-9V.B. coahuilensis 7.95 1.08 0.22 3.15 the number of copies of this functional gene is unknown. The E10m2-6V.B. coahuilensis 7.68 1.45 0.21 3.30 B. marisflavi and B. horikoshii type strains showed ten and eight F 12 112B_4D XIV. B. pumilus 7.35 1.70 0.31 2.23 copies of the rrn, respectively (Figure 2 and Supplementary G 5 169A_5R XIX. Staphylococcus 7.30 0.92 0.26 2.66 Figure S2, groups IX and XVII). Thus, the number of copies H 12 107_3D IX. B. marisflavi 7.28 0.25 0.19 3.64 quantified in these Bacillus type species was similar to the number I 13 178_5C XVI. B. idriensis 7.02 2.56 0.57 1.21 described in the strains isolated from the CCB (Figure 2). J 11 126_4D X. B. cereus 6.69 2.78 0.23 3.01 Homogeneity in rrn operon copy number was observed in the K 7 152A_5R I. B. aquamaris 6.48 0.22 0.16 4.33 B. sp. m2-34 (II; eight copies) and B. sonorensis (XI; six copies) L 8 44_1T XVII. B. horikoshii 5.20 4.12 0.19 3.64 groups. However, considerable heterogeneity and intraspecific M 10 315_11T IX. B. marisflavi 4.44 1.44 0.12 5.77 variation were observed in several other groups: B. aquamaris (I; N 11 144b_14T XIV. B. pumilus 3.67 2.79 0.11 6.30 seven to nine copies), B. sp. NRRLB-14911 (IV; eight, nine, eleven O 6 108 XII. B. atrophaeus 3.38 2.06 0.08 8.66 and twelve copies), B. vietnamiensis (VIII; eight to ten copies), P 11 127B_4D XVII. B. horikoshii 3.22 1.70 0.09 7.70 Bsubtilis(XIII; seven to eleven copies), B. pumilus (XIV; eight, A = maximum biomass reached [Ln(CFU/CFU(t = 0))]; λ = lag phase; eleven, and twelve copies) and B. horikoshii (XVII; eight, nine and µmax = maximum specific growth rate; td = doubling (generation) time. eleven copies). In addition, the phylogenetic groups composed of only one isolate showed different numbers of rrn operon copies: B. endophyticus (III; seven copies), B. sp. SP61 (VI; ten copies), reached (A). Component 2 explained 26.87% of the variance, B. sp. BA-117 (VII; nine copies), B. altitudinis (XV; eight copies), and was principally defined by adaptation time (λ)andtherrn B. idriensis (XVI; 13 copies) and B. nanhaiensis (XVIII; six copies; operon copies (Supplementary Figure S4). Meanwhile, isolates Figure 2). The Staphylococcus isolate had five copies of the rrn. with the highest copy numbers showed greater dispersion, having the most extreme parameter values (Table 1). These results Growth Parameters may indicate that the rrn operon copy number in the Bacillus Growth parameters were estimated for a subsample of 15 Bacillus from the CCB may be related to the integrated suite of growth isolates from the CCB representative of the phylogenetic diversity dynamics parameters, but not exclusively to the growth rate. present and the range of rrn operon copy numbers observed. Then, the heterogeneity in the growth dynamics of the isolates of We also characterized the isolate related to the Staphylococcus Bacillus from the CCB could be a response to the low availability genus (Table 1). Not surprisingly, given the large diversity in of nutrients and the competitive cost that represents the high this genus within the CCB, the results show a high heterogeneity number of copies of the rrn operon. in the growth parameters estimated. In agreement with these results, the lag phase period of these Bacilli is variable and it is P and N Contents and N:P Ratios in the not related with their growth rate (μmax; Supplementary Figure S3A). In addition, the maximum biomass reached was correlated Exponential Phase with the maximum specific growth rate (μmax; Supplementary To assess potential eco-physiological implications associated with Figure S3B). the oligotrophic conditions of the CCB regarding the genus Interestingly, an exploratory analysis showed no overall Bacillus, biomass C, N, and P contents were estimated during the correlation between the number of copies of the rrn operon exponential phase of growth for all strains (Supplementary Table and the growth parameters estimated (Supplementary Table S1). S2). All the isolates showed a relatively low but variable P (%) However, an arbitrary categorization of this copy number (where content (Mean = 0.496; SD = 0.616; Median = 0.258). While “low” was from five to seven copies, “mid” was from eight to C (%) and N (%) content also showed high variability among ten copies and “high” was from 11 to 14 copies) showed that the isolates [N (%): Mean = 7.14; SD = 5.61; Median = 5.49; the isolates with the fewest copies had lower levels of dispersion C(%):Mean= 64.64; SD = 26.63; Median = 59.63]. Both the in their growth parameters (Figure 3). Principal component C:N ratio (Mean = 13.18; SD = 7.18; Median = 12.32) and the analysis (PCA) was performed to describe the influence of the N:P ratio (Mean = 126.7; SD = 235.1; Median = 52.1) showed various growth parameters in these categories in the isolates from a considerable range (Table 2). Finally, the N:P ratios estimated the CCB (Figure 3). It seems that 42.71% of the variance was in the Bacillus from the CCB were substantially higher than the explained by Component 1, which was defined by doubling time ratios reported for other Bacilli (B. subtilis 10.6; Loladze and Elser, μ (td), maximum growth rate ( max) and the maximum biomass 2011).

Frontiers in Microbiology | www.frontiersin.org 7 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

(B. sonorensis and B. atrophaeus, respectively). Meanwhile, isolate A(14rrn operon copies; B. cereus) had the lowest P content of all the isolates analyzed; this isolate also had the second- highest N content. The isolates D and E (B. coahuilensis;nine and ten rrn operon copies, respectively) showed low P contents and had the lowest N content. Moreover, the previous isolates with low rrn operon copies (B, G, and O) showed low N:P ratios, while isolate A, with a high number of rrn operon copies had the highest estimated N:P ratio. In addition, the two isolates related to B. coahuilensis showed intermediate values of this elemental ratio (Table 2). These results were not consistent with the GRH and it seems that the isolates with low number of copies of the rrn operon may cope better in this oligotrophy of the CCB; although the number of operon copies quantified is high in comparison with other bacterial groups that live in other oligotrophic environments (e.g., cyanobacteria; Fegatella et al., 1998).

FIGURE 3 | Principal component analysis (PCA) of the growth DISCUSSION parameters estimated in the Bacillus isolates from the CCB. The names of isolates (letters, from A to P) were in agreement with the maximum biomass The general objectives of this work were to assess the variability reached (A)inTable 1. Color labels represent the isolates of the arbitrary of a particular ecological trait, the rrn operon copy number categorization of the number of copies of the rrn operon (upper left box), where red labels are the isolates with “low copy numbers” (five to seven), in Bacillus strains isolated from extremely oligotrophic aquatic green labels are the isolates with “intermediate copy numbers” (8 to 10) and ecosystems, and to evaluate whether there is any association black labels are the isolates with “high copy numbers” (from 11 to 14). between the variation of this trait and strain physiology. We analyzed 71 isolates of this ecosystem and found considerable variation in the ribosomal operon copy number with a tendency Despite the growth rate not being related to cellular P content toward an intermediate number of rrn operon copies. We also (Figure 4A), the correlation between rrn operon copy number documented variation in growth rate dynamics and elemental and P (%) content is negative and significant (Figure 4B). Isolate composition. While we did observe physiological associations G, with a low copy number (five copies; Staphylococcus), had the consistent with the GRH (e.g., the isolate with the slower- highest cellular content of both elements. In addition, isolates growth showed the lowest P content, and a high ratio of BandO,withsixrrn operon copies, had high P-content levels N:P). However, there were no consistent associations between copy number and growth parameters. Instead, it is likely that a variety of genomic strategies beyond variation in rrn copy TABLE 2 | C:N and N:P ratio during the exponential phase of growth in the number are employed to modulate growth in this clade of Bacillus isolates of from the CCB. Bacillus, potentially allowing for their coexistence in different rrn Isolate Phylogenetic C:N N:P niches. copies group Phylogenetic Clustering of Bacillus A 14 155B_5T X. B. cereus 4.94 965.38 B 6 118_4C XI. B. sonorensis 19.28 10 Isolates C 8 m3-18 II. B. sp. m2-34 8.35 286.47 Our phylogenetic reconstruction showed a large diversity of D9 m2-9 V.B. coahuilensis 24.82 79.09 species with isolates obtained from different habitats generally E10 m2-6 V.B. coahuilensis 18.18 52.91 widespread in the tree. This suggests that each sampling site, F 12 112B_4D XIV. B. pumilus 18.77 194.61 as well as the overall system, contains several coexisting taxa. G 5 169A_5R XIX. Staphylococcus 5.75 25.83 The microbial diversity in the Churince ecosystem has been H 12 107_3D IX. B. marisflavi 8.87 19.55 deeply documented, particularly its Bacillus population, yielding I 13 178_5C XVI. B. idriensis 17.85 40.67 55 thermo-resistant strains and several extremely halotolerant J 11 126_4D X. B. cereus 24.73 67.41 strains (Cerritos et al., 2010). Moreover, endemic Bacillus strains K 7 152A_5R I. B. aquamaris 0.62 72.58 have been described and genotyped. For instance, the genomes L 8 44_1T XVII. B. horikoshii 13.79 4.61 of B. coahuilensis and Bacillus m3-13 show several interesting M 10 315_11T IX. B. marisflavi 16.7 82.72 low-nutrient adaptations (Alcaraz et al., 2008, 2010), as well N 11 144b_14T XIV. B. pumilus 6.59 50.75 as an ancient ancestry (Moreno-Letelier et al., 2011). Further O 6 108 XII. B. atrophaeus 10.77 23.6 sampling of pond sediments in Churince led to a demonstration P 11 127B_4D XVII. B. horikoshii 10.85 51.32 of how antagonistic interactions between the Bacilli contribute Mean ± SD 13.18 ± 7.18 126.72 ± 235.13 to the large observed diversity, while maintaining a large local

Frontiers in Microbiology | www.frontiersin.org 8 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

FIGURE 4 | Relationship between the maximum specific growth rate (μmax) (A) and the number of copies of the rrn operon (B), along P (%) content, during the exponential phase. The isolates names (letters, from A to P) are within the circles in black and were in agreement with the maximum biomass reached (A) in Table 1. differentiation via either resistance or avoidance as in a paper- Previous work has described the genomic properties of some rock-scissors model (Pérez-Gutiérrez et al., 2013; Aguirre-von- Bacillus isolated from the CCB (Alcaraz et al., 2008, 2010). For Wobeser et al., 2014). example, the genome analysis of B. coahuilensis documented nine Most of our isolates came from Churince, whose large rrn operon copies (Alcaraz et al., 2010; unpublished results). terminal lake is now mostly dry due to water overexploitation However, in our analysis, we quantified eight copies, perhaps (Souza et al., 2006, 2012). Interestingly, several times during because the differences in size among operons were too small this sampling period, we recovered the same phylogroups in the to be detected via the pulse field method. Nevertheless, such sediments in the same sampling sites. It has been argued that discrepancies are common when these types of data are compared sediment is the “native” habitat of Bacillus in the CCB because (Vishnivetskaya et al., 2009). Bacillus groups isolated from the many phylogenetic groups coexist there and can be recovered CCB showed from six to 14 rrn operon copies, which is consistent consistently at different sampling times (Pérez-Gutiérrez et al., with previous quantifications of the rrn operon (from six to 15 2013). Our results support what has been observed previously: copies reported in rrnDB, Stoddard et al., 2015). This range of the considerable levels of bacterial diversity in this basin are the operon copies is not what would be expected for isolates from outcome of complex biotic interactions within the community, the CCB because the idea is that they should match a slower (i.e., CCB’s ancient geological history, low nutrient availability, and “K-selected”) life history in an oligotrophic environment such as considerable spatial and seasonal variability in environmental the CCB. This could be the case for all of the CCB isolates related conditions (Souza et al., 2012). to B. sonorensis, a soil strain that was first described in a desert sample with similar environmental conditions to those of the Variation in rrn Operon Numbers CCB (Palmisano et al., 2001; Souza et al., 2006, 2012). To evaluate We found no simple answer regarding variations in rrn operon this further, we compared our results with data from the rrnDB copy number as a response on the part of bacteria in a low- database. B. atrophaeus strain 1942 has seven rrn operon copies, nutrient environment. This was somewhat unexpected because but as mentioned above, in our analysis, we found a related strain rrn operon copy number is a well-studied functional trait that with six copies. The rrnDB B. subtilis strains from the database has been reported to be associated with bacterial lifestyle and showed from eight to ten copies; in our analysis, we observed a represents an ecological strategy for nutrient use (Klappenbach wider range (seven to eleven copies). On the other hand, seven et al., 2000; Stevenson and Schmidt, 2004; Green et al., 2008). copies of the rrn operon were observed in the B. pumilus genome, A variety of studies have quantified the copy number of while B. pumilus relatives isolated in the CCB have eight, eleven, this operon in strains isolated from environmental samples, and twelve copies. Such intraspecific variability was evaluated including some for which some Bacillus strains were analyzed by Acinas et al., (2004) for different bacterial genomes. They (Klappenbach et al., 2000; Shrestha et al., 2007; Vieria-Silva and documented three species of Bacillus that had normal variation Rocha, 2015). However, no previous studies have focused on rrn from one to three copies. Subsequent analysis with a larger operon copy number in such a diverse group of coexisting species number of Bacillus genomes showed similar variability (Rastogi within the genus Bacillus, much less in a shared environment with et al., 2009). Accordingly, the CCB’s closely related, pumilus-like extremely low nutrient availability. Bacillus showed a similar level of intraspecific variation, from one

Frontiers in Microbiology | www.frontiersin.org 9 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics to four copies. Overall, the number of copies of the rrn operon Antolinos et al., 2011, 2012; Bren et al., 2013). Furthermore, in the genus Bacillus at the CCB shows considerable variability, estimates of maximum biomass reached can be quite variable but it does not show evidence of any clear directional change because of the influence of overall nutrient availability and from previously published values for various taxa. This could be sensitivity to the waste material that accumulates during the due to the fact that very large ranges of taxa within the Bacilli exponential phase (Buchanan et al., 1997; Chorin et al., 1997). are being selected for a wide variety of responses to cope with In addition, long doubling times are characteristic of bacteria the oligotrophic environment. For example, while some save P in in oligotrophic environments (Vieria-Silva and Rocha, 2015). their ribosomes with a slow growth rate (“K strategists”), others Indeed, bacteria from other ecosystems that are extremely may maintain a high growth capacity (“r strategists”) that is limited in terms of nutrients can achieve generation times compensated by other tactics, such as phospholipid to sulfolipid of thousands of years (Jørgensen and Boetius, 2007; Labonté substitution, the presence of high-affinity P-uptake systems, and et al., 2015). Thus, the bacterial growth properties of Bacillus small genomes (Alcaraz et al., 2008; Moreno-Letelier et al., 2011). that live in the extremely oligotrophic ecosystems of the CCB likely involve a complex response to environmental rrn and nutritional conditions acting in concert with genomic Growth Parameters and Operon Copy potential. Number The analysis of bacterial growth is considered to be an rrn important tool in understanding and characterizing an organism Operon Copy Number and because it describes potential bacterial response to changes Phosphorus Availability in the CCB in environmental conditions, as well as ecological responses As previously mentioned, ecosystems in the CCB are to other microorganisms (Monod, 1949; Neidhardt, 1999). As characterized by very low P availability in water, soil, and mentioned in the results section, the growth dynamics of sediments (Elser et al., 2005; Peimbert et al., 2012), a condition Bacillus inhabitants of the CCB showed a high degree of that makes its high variation in rrn operon copy number variability in various parameters. This heterogeneity in growth somewhat surprising because we expected that such habitats rate has been previously observed for environmental strains would be dominated by taxa with low operon copy numbers. with different numbers of rrn operons (Dethlefsen and Schmidt, Previous work has suggested that rrn operon copy number is 2007). In addition, several of the estimated parameters were related to nutrient availability and especially with P because the similar to those described in other Bacillus strains under copy number is linked to the growth rate, which is associated extreme experimental growth conditions (Supplementary Table with the production of P-rich rrn (Elser, 2003; Jeyasingh and S3); however, many of these previous studies were performed Weider, 2007). Indeed, it has been shown that bacteria that with model or economically important species. Previous work live in oligotrophic environments do tend to have very low has considered the ecological importance of the rrn operon rrn operon copy numbers (< two copies; Fegatella et al., 1998; copy numbers in bacterial adaptation to different environmental Strehl et al., 1999; Lauro et al., 2009). In theory, multiple conditions (Elser et al., 2000; Klappenbach et al., 2000; Shrestha copies could allow a growth rate advantage when resources et al., 2007; Green et al., 2008). Variation in the number are abundant but would impose a competitive cost when of rrn operon copies is potentially related to the bacterial resources are limited due to the costs of the over-production growth rate because of the need to sustain high levels of rRNA of rrn (Weider et al., 2005; Dethlefsen and Schmidt, 2007; synthesis (Codon et al., 1995; Nanamiya et al., 2010; Yano Jeyasingh and Weider, 2007).WhilewedetectedmanyBacillus et al., 2013). However, in our analysis, rrn operon copy number isolates with six rrn operon copies, even this number is was not correlated with the estimated growth rate parameters. relatively high in comparison with other bacterial lineages Instead, the growth dynamics of the Bacillus from the CCB that live under low nutrient conditions (Fegatella et al., 1998; may be dictated by a combination of different physiological Strehl et al., 1999; Lauro et al., 2009). Thus, it seems that responses that are uncoupled from the rrn operon copies, such the number of copies of the rrn operon is a functional trait as differences in transcription rates or intra-cellular allocation related with the evolutionary history of the genus Bacillus,and processes. defines its ecological versatility and adaptability to different Among the various growth dynamics of the strains from environmental conditions (Klappenbach et al., 2000; Feldgarden the CCB, we observed not only that many parameters were et al., 2003; Stevenson and Schmidt, 2004; Connor et al., out of range when compared with those estimated for other 2010). previously studied Bacillus but also that they presented high A simple explanation of this incongruity is that the genus variability that at first inspection, does not seem to relate Bacillus does not pay for the full cost of high copy number under either to evolutionary history, isolation site, or rrn operon nutrient limitations due to its ability to escape from scarcity by copy number. We hypothesize that the oligotrophic condition forming spores and then germinating under better conditions. in the CCB may have had a significant effect on the Indeed, stress response capacity has been shown to be related Bacillus growth dynamics that contribute to this variability. to rrn operon multiplicity (Nanamiya et al., 2010; Yano et al., For example, long adaptation times are related to stressful 2013). However, spore formation in Bacillus of the CCB is not conditions affecting the speed of bacterial growth, like limited a given, due to the loss of many of the genes of the spore- nutrient availability (Chorin et al., 1997; Schaechter, 2006; forming complex in the sequenced genomes (Alcaraz et al., 2010)

Frontiers in Microbiology | www.frontiersin.org 10 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics and the difficulty of obtaining spores experimentally in all the persist in this oligotrophic ecosystem. Analogous strategies have isolated strains. Nevertheless, B. coahuilensis m4-4 and B. sp. m3- been described in other organisms whose growth parameters are 13 have eight and nine rrn operon copies, respectively, like other also affected by various environmental and biotic factors (Pianka, spore-forming Bacilli (B. halodurans C-125, eight rrn operon 1970; Page, 2002; Lipowsky et al., 2012). Variable lag phases copies; B. amyloliquefaciens CC178, 9 rrn operon copies). Thus, may help in adapting to changing environmental conditions to the variability in the rrn operon copy number could reflect reach optimum growth with long generation times (Crooks, 2005; broader life history strategies in which each taxa uses different Wangen and Webster, 2006; Daehler, 2009). We also note that the sets of resources or inhabits distinct microhabitats in structured retardation of growth is a common result of intense interaction sediments, potentially decreasing competition. In agreement with with other organisms, as well as stressful conditions (Gao et al., this view, various bacterial communities have been shown to 2013; Tsugama et al., 2014). To date, these ecological strategies have similar functional heterogeneity regarding other important in bacteria have been largely related only to nutrient availability ecological traits (Martínez-Alonso et al., 2004; Giovannoni and (Klappenbach et al., 2000; Fierer et al., 2007; Shrestha et al., Stingl, 2005; Martiny et al., 2006). Additional work is needed to 2007). However, the intensity of direct inter-specific interactions understand the importance of conserving high rrn operon copy (such as chemical antagonisms) can also establish coexistence, numbers in CCB Bacillus. and these interactions are known to be particularly intense in Our overall findings about diversity in Bacillus rrn operon the CCB (Souza et al., 2012; Pérez-Gutiérrez et al., 2013; Aguirre- number in the CCB do not seem to conform to the broader von-Wobeser et al., 2014). These inferences suggest a bet-hedging context of the GRH (Elser et al., 2000; Elser, 2006). For example, strategy on the part of CCB bacteria in which expression of RNA isolate A (B. cereus) had the highest number of copies of the rrn genes is tightly controlled due to low P-conditions (reflected operon (14) and also had the slowest growth rate and highest in their overall low P content and high N:P ratio), but when N:P ratio (965) when growing on media produced using the chemical antagonism is successful, resources suddenly arrive, and CCB’s natural waters. Furthermore, several isolates with low rrn the rrn operons are activated to grow rapidly under the nutrient operon numbers showed some of the highest P-content values, bounty. contrary to the GRH. However, the high N:P ratio and slow The main results of our work indicate that the rrn operon growth rate of isolate A is consistent with the development of copy number exhibits considerable variation among field-isolated severe P limitations for this high-copy-number strain, resulting Bacilli and that considerable variation also exist in their growth in its high biomass N:P ratio. Then, it seems that the isolates properties and chemical composition. However, rrn operon with lower number of copies of the rrn operon may cope better copy number appears to be largely uncoupled from growth in this oligotrophy of the CCB. To more effectively test the and chemical properties in this clade. Further investigation genetic dimension of the GRH, each strain would need to be is needed to understand the ecological and physiological raised under optimal conditions at its genetically constrained importance of this rrn operon variability, as mediated by gene maximal growth rate. Indeed, this inference is supported by transcriptional regulation, and its influence on ribosome and the higher dispersion of growth parameters seen for high- protein content and thus N:P stoichiometry (Gourse et al., copy-number strains. That is, low-copy-number strains may 1996; Fegatella and Cavicchioli, 2000; Dethlefsen and Schmidt, have a limited range of growth variation, regardless of media, 2007; Scott et al., 2010; Piir et al., 2011). It may be that while high-copy-number strains may have a considerable range the extreme oligotrophic conditions in the CCB have imposed of growth, depending on whether or not the environment is important physiological constraints on resource allocation and well-matched to their needs. Another possible explanation for growth rate, as well as the expression of the rRNA genes, the decoupling of rrn operon number from growth rate and and thus, the rate of production of ribosomes per rrn operon stoichiometric properties is that CCB Bacillus are selected for copy differs considerably among strains. More detailed studies, fine-tuned signaling with resource supplies or for variation in the including competition experiments involving Bacillus strains rates of rRNA genes expression, thus disconnecting copy number isolated from different CCB environments and subject to various from RNA production and growth rate (i.e., low-copy-strains environmental limitations (such as differences in nutrient supply may have high levels of transcription for each copy, while high- concentrations, ratios, and supply schedules), may be needed copy strains may more stringently express each copy). In any to identify the ecological and evolutionary significance of rrn case, our data do not provide a clear resolution regarding the operon copy number variation among microbes in the habitats validity of the genetic components of the GRH within the Bacillus of Cuatro Ciénegas and similar nutrient-deficient habitats. of the CCB. It is possible that the consideration of a broader range of bacterial taxa, as well as the more extensive testing of growth conditions, are needed in order to more rigorously test the AUTHOR CONTRIBUTIONS GRH in the microbial realm, using approaches that can overcome the possible impacts of physiological conditions, phylogenetic JV-A: Primary author, experimental design, amplification inertia, and taxon-specific lifecycle strategies (e.g., sporulation) and analysis of genetic material, PFGE, analysis of data. in terms of confounding the interpretations. LE-F: Experimental design, analysis of data. GD-S: PFGE We suggest that the growth patterns described in the Bacillus standardization, analysis of data. PM-Z: Growth curves isolates from the CCB, as well as the high variability in rrn operon standardization, parameter estimations. JG-P: Statistical analysis. copy number, represent ecological strategies that allow them to JL: Cell contents of phosphorus (P) and nitrogen (N). JE:

Frontiers in Microbiology | www.frontiersin.org 11 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

Experimental design, analysis of data. GO-A: Experimental SUPPLEMENTARY MATERIAL design, analysis of data. VS: Group leader, experimental design, analysis of data. The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.01486 ACKNOWLEDGMENTS FIGURE S1 | Growth conditions assays and Gompertz growth curves of the Bacillus isolates from the CCB. (A) Different nutritional conditions were Thanks are due to Posgrado en Ciencias Biológicas of the analyzed to define the optimum conditions and thus describe the growth Universidad Nacional Autónoma de México (UNAM). This dynamics of the Bacillus:A= Marine broth (DifcoTM 2216); B = CCB water; work is the doctoral research of JV-A in the Doctorado en C = CCB water + P(KH2PO4, 5 g per liter); and D = CCB water + N (tryptone, Ciencias Biológicas program (CVU: 216032; CONACyT fellow 5 g per liter). Growth was quantified via culture turbidity (OD600; BioPhotometer number: 207187). We would like to thank Ariadnna del C. Cruz- Plus, Eppendorf) after 12 h of growth, and the data were normalized to define the optimal conditions for growth. (B) Growth curves were described after linear Córdoba and Juan Xicohtencatl-Cortés of the Laboratorio de regression analysis according to the Gompertz equation. Isolate names were in Bacteriología Intestinal (Hospital Infantil de México “Federico agreement with the maximum growth reached (A), followed by the number of Gómez”), and José Luis Méndez and Rosario Morales of the copies of the rrn operon for every isolate. Growth curves were plotted with the Laboratorio de Genómica Bacteriana (UNAM), because of their data of the plate count assays [Ln(CFU/CFU(t = 0))]. technical assistance in PFGE standardization, Africa Islas of the FIGURE S2 | rRNA operon copy number in the genus Bacillus isolated in Laboratorio de Bacteriología Molecular (CINVESTAV-Unidad the CCB. For every isolate, the separation of fragments with PFGE is shown on Irapuato) for providing the strains needed to perform this work, the left and Southern-blot confirmation is shown on the right. At the bottom of the Felipe García-Oliva (Biogeoquímica de Suelos; CIEco, UNAM) figure is the total number of rRNA operon copies quantified. The red asterisk represents the fragments identified with hybridization analysis. and Mario Soberón-Chávez (Departamento de Microbiología Molecular; IBT, UNAM) for their advice and input of ideas FIGURE S3 | Relationship between the growth parameters estimated in Bacillus λ μ μ throughout the development of this work, Ana Gutiérrez- the isolates from the CCB. (A) vs max and, (B) max vs A. Isolate names (letters, from A to P) are within the circles in black and were in agreement Preciado (Institut Cavanilles de Biodiversitat i Biologia Evolutiva, with the maximum biomass reached (A) in Table 1.A= maximum biomass Universitat de València) for her comments and observations that reached [Ln(CFU/CFU(t = 0))]; λ = lag phase; μmax = maximum specific growth contributed to the improvement of the manuscript. Grants from rate. Alianza WWF-Fundación Carlos Slim to VS and LE-F and U.S. FIGURE S4 | Relative contribution (%) per variable in the PCA of the NSF (DEB-0950179) to JE supported this study. Laura Espinosa- growth parameters estimated in the Bacillus isolates from the CCB. = λ = = Asuar and Erika Aguirre-Planter helped in the general technical A maximum growth reached; lag phase; td doubling time; and μ = logistics during the entire project. max maximum specific growth rate.

REFERENCES APHA (2005). Standard Methods for the Examination of Water and Wastewater. Washington, DC: American Publish Health Association. Acinas, S. G., Marcelino, L. A., Klepac-Ceraj, V., and Polz, M. F. (2004). Bren, A., Hart, Y., Dekel, E., Koster, D., and Alon, U. (2013). The last generation of Divergence and redundancy of 16S rRNA sequences in genomes with multiple bacterial growth in limiting nutrient. BMC Syst. Biol. 7:27. doi: 10.1186/1752- rrn operons. J. Bacteriol. 186, 2629–2635. doi: 10.1128/JB.186.9.2629-263 0509-7-27 5.2004 Buchanan, R. L., Whiting, R. C., and Damert, W. C. (1997). When in simple Aguirre-von-Wobeser, E., Soberón-Chávez, G., Eguiarte, L. E., Ponce-Soto, G. Y., good enough: a comparison of the Gompertz, Baranyi, and three-phase linear Vázquez-Rosas-Landa, M., and Souza, V. (2014). Two-role model of an models for fitting bacterial growth curves. Food Microbiol. 14, 313–326. doi: interaction network of free-living γ-proteobacteria from an oligotrophic 10.1006/fmic.1997.0125 environment. Environ. Microbiol. 16, 1366–1377. doi: 10.1111/1462-2920. Burdett, I. D. J., Kirkwood, T. B. L., and Whalley, J. B. (1986). Growth kinetics 12305 of individual Bacillus subtilis cells and correlation with nucleoid extension. Alcaraz, L. D., Moreno-Hagelsieb, G., Eguiarte, L. E., Souza, V., Herrera-Estrella, L., J. Bacteriol. 167, 219–230. and Olmedo, G. (2010). Understanding the evolutionary relationships and Cavicchioli, R., Ostrowski, M., Fegatella, F., Goodchild, A., and Guixa- major traits of Bacillus through comparative genomics. BMC Genomics 11:332. Boixereu, N. (2003). Life under nutrient limitation in oligotrophic doi: 10.1186/1471-2164-11-332 marine environments: an eco/physiological perspective of Sphingopyxis Alcaraz, L. D., Olmedo, G., Bonilla, G., Cerritos, R., Hernández, G., Cruz, A., alaskensis (formerly Sphingophyxis alaskensis). Microb. Ecol. 45, et al. (2008). The genome of Bacillus coahuilensis reveals adaptations 203–217. essential for survival in the relic of an ancient marine environment. Cerritos, R., Eguiarte, L. E., Avitia, M., Siefert, J., Travisano, M., Rodríguez- Proc. Natl. Acad. Sci. U.S.A. 105, 5803–5808. doi: 10.1073/pnas.0800 Verdugo, A., et al. (2010). Diversity of culturable thermo-resistant aquatic 981105 bacteria along an environmental gradient in Cuatro Ciénegas, Coahuila, Antolinos, V., Muñoz, M., Ros-Chumillas, M., Aznar, A., Periago, P. M., and México. Antonie Van Leeuwenhoek 98, 1–16. doi: 10.1007/s10482-010- Fernández, P. S. (2011). Combined effect of lysozyme and nisin at different 9490-9 incubation temperature and mild heat treatment on the probability of time Cerritos, R., Vinuesa, P., Eguiarte, L. E., Herrera-Estrella, L., Alcaraz-Peraza, growth of Bacillus cereus. Food Microbiol. 28, 305–310. doi: 10.1016/j.fm.2010. L. D., Arvizu-Gómez, J. L., et al. (2008). Bacillus coahuilensis sp. nov., a 07.021 moderately halophilic species from a desiccated lagoon in the Cuatro Cienegas Antolinos, V., Muñoz-Cuevas, M., Ros-Chumillas, M., Periago, P. M., Fernández, valley in Coahuila, Mexico. Int. J. Syst. Evol. Microbiol. 58, 919–923. doi: P. S., and Marc, Y. L. (2012). Modelling the effects of temperature and osmotic 10.1099/ijs.0.64959-0 shifts on the growth kinetics of Bacillus weihenstephanensis in broth and food Chorin, E., Thuault, D., Cléret, J. J., and Bourgeois, C. M. (1997). Modeling products. Int. J. Food Microbiol. 158, 36–44. doi: 10.1016/j.ijfoodmicro.2012. Bacillus cereus growth. Int. J. Food Microbiol. 38, 229–234. doi: 10.1016/S0168- 06.017 1605(97)00110-4

Frontiers in Microbiology | www.frontiersin.org 12 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

Chubukov, V., and Sauer, U. (2014). Environmental dependence of Green, J. L., Bohannan, B. J. M., and Whitaker, R. J. (2008). Microbial stationary-phase metabolism in Bacillus subtilis and Escherichia biogeography: from taxonomy to traits. Science 320, 1039–1042. doi: coli. Appl. Environ. Microbiol. 80, 2901–2909. doi: 10.1128/AEM. 10.1126/science.1153475 00061-14 Hughes, A. R., Inouye, B. D., Johnson, M. T. J., Underwood, N., and Vellend, M. Codon, C., Liveris, D., Squires, C., Schwartz, I., and Squires, C. L. (1995). rRNA (2008). Ecological consequences of genetic diversity. Ecol. Lett. 11, 609–623. doi: operon multiplicity in Escherichia coli and the physiological implications of rrn 10.1111/j.1461-0248.2008.01179.x inactivation. J. Bacteriol. 177, 4152–4156. Jeyasingh, P. D., and Weider, L. J. (2007). Fundamental links between Collins, J. F., and Richmound, M. H. (1962). Rate of increase in length of bacteria genes and elements: evolutionary implications of ecological between divisions. J. Gen. Microbiol. 28, 15–33. doi: 10.1099/00221287-2 stoichiometry. Mol. Ecol. 16, 4649–4661. doi: 10.1111/j.1365-294X.2007. 8-1-15 03558.x Connor,N.,Sikorski,J.,Rooney,A.P.,Kopac,S.,Koeppel,A.F.,Burger,A.,etal. Jørgensen, B. B., and Boetius, A. (2007). Feast and famine-microbial life in (2010). Ecology of speciation in the genus Bacillus. Appl. Environ. Microbiol. 76, the deep-sea bed. Nat. Rev. Microbiol. 5, 770–781. doi: 10.1038/nrmicro 1349–1358. doi: 10.1128/AEM.01988-09 1745 Crooks, J. A. (2005). Lag times and exotic species: the ecology and management Kacena, M. A., Merrell, G. A., Manfredi, B., Smith, E. E., Klaus, D. M., and Todd, P. of biological invasions in slow-motion. Ecoscience 12, 316–329. doi: (1999). Bacterial growth in space flight: logistic growth curve parameters for 10.2980/i1195-6860-12-3-316.1 Escherichia coli and Bacillus subtilis. Appl. Microbiol. Biotechnol. 51, 229–234. Daehler, C. C. (2009). Short lag times for invasive tropical plants: evidence doi: 10.1007/s002530051386 from experimental planting in Hawaii. PLoS ONE 4:e4462. doi: Klappenbach, J. A., Dunbar, J. M., and Schmidt, T. M. (2000). rRNA operon copy 10.1371/journal.pone.0004462 number reflects ecological strategies of bacteria. Appl. Environ. Microbiol. 66, Delgado, G., Souza, V., Morales, R., Cerritos, R., González-González, A., Méndez, 1328–1333. doi: 10.1128/AEM.66.4.1328-1333.2000 J. L., et al. (2013). Genetic characterization of atypical Citrobacter freundii. PLoS Klappenbach, J. A., Saxman, P. R., Cole, J. R., and Schmidt, T. M. (2001). rrndb: the ONE 8:e74120. doi: 10.1371/journal.pone.0074120 ribosomal RNA operon copy number database. Nucleic Acids Res. 29, 181–184. Dethlefsen, L., and Schmidt, T. M. (2007). Performance of the translational doi: 10.1093/nar/29.1.181 apparatus varies with the ecological strategies of bacteria. J. Bacteriol. 189, Labonté, J. M., Field, E. K., Lau, M., Chivian, D., Van Heerden, E., Wommack, 3237–3245. doi: 10.1128/JB.01686-06 K. E., et al. (2015). Single cell genomics indicates horizontal gene transfer and Elser, J. J. (2003). Biological stoichiometry: a theoretical framework connecting viral infections in a deep subsurface Firmicutes population. Front. Microbiol. ecosystem ecology, evolution, and biochemistry for application in 22:349. doi: 10.3389/fmicb.2015.00349 astrobiology. Int. J. Astrobiol. 2, 185–193. doi: 10.1017/S147355040 Lauro,F.M.,McDougald,D.,Thomas,T.,Williams,T.J.,Egan,S.,Rice,S., 3001563 et al. (2009). The genomic basis of trophic strategy in marine bacteria. Elser, J. J. (2006). Biological stoichiometry: a chemical bridge between ecosystem Proc. Natl. Acad. Sci. U.S.A. 106, 15527–15533. doi: 10.1073/pnas.09035 ecology and evolutionary biology. Am. Nat. 168, S25–S35. doi: 10.1086/5 07106 09048 Lipowsky, A., Roscher, C., Schumacher, J., and Schmid, B. (2012). Density- Elser, J. J., Sterner, R. W., Gorokhova, E., Fagan, W. F., Markow, T. A., Cotner, J. B., independent mortality and increasing plant diversity are associated with et al. (2000). Biological stoichiometry from genes to ecosystems. Ecol. Lett. 3, differentiation of Taraxacum officinale into r-and K-strategists. PLoS ONE 540–550. doi: 10.1046/j.1461-0248.2000.00185.x 7:e28121. doi: 10.1371/journal.pone.0028121 Elser, J. J., Watts, J., Schampel, J. H., and Farmer, J. (2005). Early Cambrian Liu, S., Hessel, A., and Sanderson, K. E. (1993). Genomic mapping with I-CeuI, food webs on a trophic knife-edge? A hypothesis and preliminary data an intron-encoded endonuclease specific for genes for ribosomal RNA, in from a modern stromatolite-based ecosystem. Ecol. Lett. 9, 295–303. doi: Salmonella spp., Escherichia coli, and other bacteria. Proc. Natl. Acad. Sci. U.S.A. 10.1111/j.1461-0248.2005.00873.x 90, 6874–6878. doi: 10.1073/pnas.90.14.6874 Fegatella, F., and Cavicchioli, R. (2000). Physiological responses to starvation Loladze, I., and Elser, J. J. (2011). The origins of the Redfield nitrogen-to- in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain phosphorus ratio are in a homoeostatic protein-to-rRNA ratio. Ecol. Lett. 14, RB2256. Appl. Environ. Microbiol. 66, 2037–2044. doi: 10.1128/AEM.66.5.2037- 244–250. doi: 10.1111/j.1461-0248.2010.01577.x 2044.2000 Maciá, E. (2005). The role of phosphorus in chemical evolution. Chem. Soc. Rev. Fegatella, F., Lim, J., Kjelleberg, S., and Cavicchioli, R. (1998). Implications 34, 691–701. doi: 10.1039/b416855k of rRNA operon copy number and ribosome content in the marine Martínez-Alonso, M., Mir, J., Caumette, P., Gaju, N., Guerrero, R., and oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Esteve, I. (2004). Distribution of phototrophic populations and primary Environ. Microbiol. 64, 4433–4438. production in a microbial mat from the Ebro Delta, Spain. Int. Microbiol. 7, Feldgarden, M., Byrd, N., and Cohan, F. M. (2003). Gradual evolution in bacteria: 19–25. evidence from Bacillus systematics. Microbiology 149(Pt 12), 3565–3573. doi: Martiny, A. C., Coleman, M. L., and Chisholm, S. W. (2006). Phosphate acquisition 10.1099/mic.0.26457-0 genes in Prochlorococcus ecotypes: evidence for genome-wide adaptation. Fierer, N., Brandford, M. A., and Jackson, R. B. (2007). Toward an ecological Proc. Natl. Acad. Sci. U.S.A. 103, 12552–12557. doi: 10.1073/pnas.06013 classification of soil bacteria. Ecology 88, 1354–1364. doi: 10.1890/0 01103 5-1839 Martiny, A. C., Huang, Y., and Li, W. (2009). Occurrence of phosphate Gao, R., Wan, Z. Y., and Wong, S. M. (2013). Plant growth retardation acquisition genes in Prochlorococcus cells from different ocean regions. and conserved miRNAs are correlated to hibiscus chlorotic ringspot Environ. Microbiol. 11, 1340–1347. doi: 10.1111/j.1462-2920.2009. virus infection. PLoS ONE 8:e85476. doi: 10.1371/journal.pone.00 01860.x 85476 McGill, B. J., Enquist, B. J., Weiher, E., and Westoby, M. (2006). Rebuilding Giovannoni, S. J., and Stingl, U. (2005). Molecular diversity and ecology community ecology from functional traits. Trends Ecol. Evol. 21, 178–185. doi: of microbial plankton. Nature 437, 343–348. doi: 10.1038/nature 10.1016/j.tree.2006.02.002 04158 Membré, J. M., Leporq, B., Vialette, M., Mettler, E., Perrier, L., Thuault, D., Goto, K., Omura, T., Hara, Y., and Sadaie, Y. (2000). Application of the et al. (2005). Temperature effect on bacterial growth rate: quantitative partial 16S rDNA sequence as an index for rapid identification of species approach including cardinal values and variability estimates to perform in the genus Bacillus. J. Gen. Appl. Microbiol. 46, 1–8. doi: 10.2323/ growth simulations on/in food. Int. J. Food Microbiol. 100, 179–186. doi: jgam.46.1 10.1016/j.ijfoodmicro.2004.10.015 Gourse, R. L., Gaal, T., Bartlett, M. S., Appleman, J. A., and Ross, W. Monod, J. (1949). The growth of bacterial cultures. Annu.Rev.Microbiol.3, (1996). rRNA transcription and growth rate-dependent regulation of ribosome 371–394. doi: 10.1146/annurev.mi.03.100149.002103 synthesis in Escherichia coli. Annu. Rev. Microbiol. 50, 645–677. doi: Moreno-Letelier, A., Olemdo, G., Eguiarte, L. E., Martinez-Castilla, L., and 10.1146/annurev.micro.50.1.645 Souza, V. (2011). Parallel evolution and horizontal gene transfer of the

Frontiers in Microbiology | www.frontiersin.org 13 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

pst operon in Firmicutes from oligotrophic environments. Int. J. Evol. Biol. Souza, V., Siefert, J. L., Escalante, A. E., Elser, J. J., and Eguiarte, L. E. (2012). The 2011:781642. doi: 10.4061/2011/781642 Cuatro Ciénegas Basin in Coahuila, Mexico: an astrobiological Precambriam Nakasone, K., Noriaki, M., Yoshihiro, T., Rumie, S., Go, M., Tokuki, S., et al. (2000). park. Astrobiology 12, 641–647. doi: 10.1089/ast.2011.0675 Characterization and comparative study of the rrn operons of alkaliphilic Spencer, C. C., Tyerman, J., Bertrand, M., and Doebeli, M. (2008). Adaptation Bacillus halodurans C-125. Extremophiles 4, 209–214. doi: 10.1007/PL00 increases the likelihood of diversification in an experimental bacterial 010713 linage. Proc.Natl.Acad.Sci.U.S.A.105, 1585–1589. doi: 10.1073/pnas.07085 Nanamiya, H., Sato, M., Masuda, K., Sato, M., Wada, T., Suzuki, S., et al. (2010). 04105 Bacillus subtilis mutants harbouring a single copy of the rRNA operon exhibit Sterner, R. W., and Elser, J. J. (2002). Ecological Stoichiometry: The Biology of severe defects in growth and sporulation. Microbiology 156, 2944–2952. doi: Elements from Molecules to the Biosphere. Princeton, NJ: Princeton University 10.1099/mic.0.035295-0 Press. Neidhardt, F. C. (1999). Bacterial growth: constant obsession with dN/dt. Stevenson, B. S., and Schmidt, T. M. (2004). Life history implications of rRNA gene J. Bacteriol. 181, 7405–7408. copy number in Escherichia coli. Appl. Environ. Microbiol. 70, 6670–6677. doi: Ng, T. M., and Schaffner, D. W. (1997). Mathematical models for the effects of pH, 10.1128/AEM.70.11.6670-6677.2004 temperature, and sodium chloride on the growth of Bacillus stearothermophilus Stoddard, S. F., Smith, B. J., Hein, R., Roller, B. R. K., and Schmidt, T. M. (2015). in salty carrots. Appl. Environ. Microbiol. 63, 1237–1243. rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and Page, C. N. (2002). Ecological strategies in fern evolution: a neopteridological archaea and a new foundation for future development. Nucleic Acids Res. 43, overview. Rev. Palaeobot. Palynol. 119, 1–33. doi: 10.1016/S0034- D593–D598. doi: 10.1093/nar/gku1201 6667(01)00127-0 Strehl, B., Holtzendorff, J., Partensky, F., and Hess, W. R. (1999). A small Palmisano, M. M., Nakamura, L. K., Duncan, K. E., Istock, C. A., and Cohan, F. M. and compact genome in the marine cyanobacterium Prochlorococcus marinus (2001). Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, CCMP 1375: lack of an intron in the gene for tRNAr (Leu)UAA and a single isolated from soil in the Sonoran Desert, Arizona. Int. J. Syst. Evol. Microbiol. copy of the rRNAop. FEMS Microbiol. Lett. 181, 261–266. doi: 10.1111/j.1574- 51, 1671–1679. doi: 10.1099/00207713-51-5-1671 6968.1999.tb08853.x Peimbert, M., Alcaraz, L. D., Bonilla-Rosso, G., Olmedo-Alvarez, G., García- Sutherland, J. P., Aherne, A., and Beaumont, A. L. (1996). Preparation and Oliva, F., Segovia, L., et al. (2012). Comparative metagenomics of two microbial validation of growth model for Bacillus cereus: the effects of temperature, pH, mats at Cuatro Ciénegas Basin I: ancient lessons on how to cope with sodium chloride and carbon dioxide. Int. J. Food Microbiol. 30, 359–372. doi: an environment under severe nutrient stress. Astrobiology 12, 648–658. doi: 10.1016/0168-1605(96)00962-2 10.1089/ast.2011.0694 Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011). Pérez-Gutiérrez, R. A., López-Ramírez, V., Islas, A., Alcaraz, L. D., Hernández- MEGA5: molecular evolutionary genetics analysis using maximum likelihood, González, I., Luna Oliviera, B. C., et al. (2013). Antagonism influences assembly evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, of a Bacillus guild in a local community and is depicted as a food-chain network. 2731–2739. doi: 10.1093/molbev/msr121 ISME J. 7, 487–497. doi: 10.1038/ismej.2012.119 Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2002). Multiple sequence Pianka, E. R. (1970). On r- and K-Selection. Am. Nat. 104, 592–597. doi: alignment using ClustalW and ClustalX. Curr. Protoc. Bioinformatics Chapter 10.1086/282697 2, Unit2.3. doi: 10.1002/0471250953.bi0203s00 Piir, K., Paier, A., Liiv, A., Tenson, T., and Maiväli, Ü (2011). Ribosome Tsugama, D., Liu, S., and Takano, T. (2014). Analysis of functions of VIP1 and its degradation in growing bacteria. EMBO Rep. 12, 458–462. doi: 10.1038/embor. close homologs in osmosensory responses of Arabidopsis thaliana. PLoS ONE 2011.47 9:e103930. doi: 10.1371/journal.pone.0103930 Powell, E. O. (1956). Growth rate and generation time of bacteria, with Valík, L., Görner, F., and Lauková, D. (2003). Growth dynamics of Bacillus cereus special reference to continuous culture. J. Gen. Microbiol. 15, 492–511. doi: and self-life of pasteurised milk. Czech J. Food Sci. 21, 195–202. 10.1099/00221287-15-3-492 Van Mooy, B. A. S., Fredricks, H. F., Pedler, B. E., Dyhrman, S. T., Karl, Prüβ, B. M., Francis, K. P., von Stetten, F., and Scherer, S. (1999). Correlation D. M., Koblízek, M., et al. (2009). Phytoplankton in the ocean use non- of 16S ribosomal DNA signature with temperature-dependent growth rates of phosphorus lipids in response to phosphorus scarcity. Nature 458, 69–72. doi: mesophilic and psychrotolerant strains of the Bacillus cereus group. J. Bacteriol. 10.1038/nature07659 181, 2624–2630. Vieria-Silva, S., and Rocha, E. P. C. (2015). The systemic imprint of growth Rastogi, R., Wu, M., Dasgupta, I., and Fox, G. E. (2009). Visualization of and its uses in ecological (Meta) genomics. PLoS Genet. 6:e1000808. doi: ribosomal RNA operon copy number distribution. BMC Microbiol. 9:208. doi: 10.1371/journal.pgen.1000808 10.1186/1471-2180-9-208 Vishnivetskaya, T. A., Kathariou, S., and Tiedje, J. M. (2009). The Exiguobacterium Ratkowsky,D.A.,Lowry,R.K.,McMeekin,T.A.,Stokes,A.N.,andChandler,R.E. genus: biodiversity and biogeography. Extremophiles 13, 541–555. doi: (1983). Model for bacterial culture growth rate troughout the entire biokinetic 10.1007/s00792-009-0243-5 temperature range. J. Bacteriol. 154, 1222–1226. Wangen, S. R., and Webster, C. R. (2006). Potential for multiple lag phases Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A during biotic invasions: reconstructing an invasion of the exotic tree Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press, Acer platanoides. J. Appl. Ecol. 43, 258–268. doi: 10.1111/j.1365-2664.2006. 31–39. 01138.x Schaechter, M. (2006). From growth physiology to system biology. Int. Microbiol. Warth, A. D. (1978). Relationship between the heat resistance of spores and the 9, 157–161. optimum and maximum growth temperatures of Bacillus species. J. Bacteriol. Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z., and Hwa, T. (2010). 134, 699–705. Interdependence of cell growth and gene expression: origins and consequences. Weider, L. J., Elser, J. J., Crease, T. J., Mateos, M., Cotner, J. B., and Science 330, 1099–1102. doi: 10.1126/science.1192588 Markow, T. A. (2005). The functional significance of ribosomal (r) Shrestha, P. M., Noll, M., and Liesack, W. (2007). Phylogenetic identity, growth- DNA variation: impacts on the evolutionary ecology of organisms. Annu. response time and rRNA operon copy number of soil bacteria indicate Rev. Ecol. Evol. Syst. 36, 219–242. doi: 10.1146/annurev.ecolsys.36.102003. different stages of community succession. Environ. Microbiol. 9, 2464–2474. doi: 152620 10.1111/j.1462-2920.2007.01364.x Whitaker, R. J., Grogan, D. W., and Taylor, J. W. (2003). Geographic barriers isolate Souza, V., Eguiarte, L. E., Siefert, J. S., and Elser, J. J. (2008). Microbial endemism: endemic populations of hyperthermophilic archaea. Science 301, 976–978. doi: does phosphorous limitation enhance speciation? Nat. Rev. Microbiol. 6, 559– 10.1126/science.1086909 564. doi: 10.1038/nrmicro1917 Xu, J. (2006). Microbial ecology in the age of genomics and metagenomics: Souza, V., Espinosa-Asuar, L., Escalante, A. E., Eguiarte, L. E., Farmer, L. E., concepts, tools, and recent advances. Mol. Ecol. 15, 1713–1731. doi: Forney, L., et al. (2006). An endangered oasis of aquatic microbial biodiversity 10.1111/j.1365-294X.2006.02882.x in the Chihuahuan desert. Proc. Natl. Acad. Sci. U.S.A. 103, 6565–6570. doi: Yano, K., Wada, T., Suzuki, S., Tagami, K., Matsumoto, T., Shiwa, Y., et al. 10.1073/pnas.0601434103 (2013). Multiple rRNAops are essential for efficient cell growth and sporulation

Frontiers in Microbiology | www.frontiersin.org 14 January 2016 | Volume 6 | Article 1486 Valdivia-Anistro et al. rrn Operon and Growth Dynamics

as well as outgrowth in Bacillus subtilis. Microbiology 159, 2225–2236. doi: Copyright © 2016 Valdivia-Anistro, Eguiarte-Fruns, Delgado-Sapién, Márquez- 10.1099/mic.0.067025-0 Zacarías, Gasca-Pineda, Learned, Elser, Olmedo-Alvarez and Souza. This is an Zwietering, M. H., Jongenburger, I., Rombouts, F. M., and van´t Riet, K. (1990). open-access article distributed under the terms of the Creative Commons Attribution Modeling of the bacterial growth cure. Appl. Environ. Microbiol. 56, 1875–1881. License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original Conflict of Interest Statement: The authors declare that the research was publication in this journal is cited, in accordance with accepted academic practice. conducted in the absence of any commercial or financial relationships that could No use, distribution or reproduction is permitted which does not comply with these be construed as a potential conflict of interest. terms.

Frontiers in Microbiology | www.frontiersin.org 15 January 2016 | Volume 6 | Article 1486